A multicenter collaboration between the NCI-Frederick Molecular Targets Discovery and HIV Drug Resistance Programs, the National Institute of Child Health and Development, and the University of Pittsburgh has used high-throughput robotics to screen several libraries, totaling 250,000 compounds, for small-molecule inhibitors of HIV RNase H function. Secondary screening against bacterial and human RNase H has addressed whether selectivity for the retroviral enzyme can be achieved. Several structural classes of RNase H inhibitors have been identified by this strategy, the most potent of which was the hydroxylated tropolone beta-thujaplicinol. Derived from the bark of the western cedar Thuja plicata, beta-thujaplicinol inhibited HIV-1 RT/RNaseH at a concentration of 0.2 uM, while the IC50 for human RNase H was 6.0 uM and that of the bacterial enzyme &gt;50 uM. In addition, beta-thujaplicinol was shown to synergize with the nonnucleoside inhibitor calanolide A, strengthening contentions from other groups that both the DNA polymerase and RNase H activities of HIV-1 RT can be simultaneously targeted. Vinylogous ureas constitute a second structural class of RNase H inhibitors, and a patent covering these inhibitors has been submitted. Structural studies to define the binding site of the most potent RNase H inhibitors are currently underway. We are continuing our studies on RNase H as an antiviral target by (1) using crystallographic data to alter residues of RT implicated in inhibitor binding, (2) synthesizing novel derivatives of both structural classes, and (3) investigating the relationship between impaired RNase H function and increased excision of chain-terminating nucleoside RT inhibitors (NRTIs). Site-specific derivatization with a novel trifunctional agent will also be investigated as a general method of creating fluorescent proteins, allowing fluorescence polarization to be used for screening protein:protein interactions. Initial studies will focus on the interaction of the host protein lens epithelium-derived growth factor (LEDGF) with HIV-1 integrase. We previously identified two classes of HIV-1 RNase H inhibitors that work by different mechanisms. The alpha-hydroxytropolone pharmacophore chelates divalent metal at the RNase H active site, exemplified by our high-resolution crystal structure of HIV-1 RT containing the nonnucleoside RT inhibitor (NNRTI) TMC278 and the natural product manicol. In contrast, vinylogous ureas occupy a site in the p51 thumb subdomain, suggesting allosteric inhibition. Studies on HIV-1 RNase H inhibitors will be extended through collaborations with the NIH Chemical Genomics Center (alpha-hydroxytropolones) and the Department of Pharmacy, University of Cagliari, Italy (vinylogous ureas), to synthesize derivatives of lead compounds with improved potency and in vivo efficacy. Preparing first-generation ATCUN-hydroxytropolone complexes as catalytic RNase H inhibitors is a new area that will complement these projects. ATCUNs are short peptide motifs that, by coordinating Cu2+ or Ni2+, can generate reactive oxygen species capable of cleaving/modifying the target biomolecule in their immediate vicinity. Additional uses of ATCUN-based metallopeptides and metallomicrobicides will be investigated. [Corresponds to Le Grice Project 2 in the October 2011 site visit report of the HIV Drug Resistance Program]